Infection of avian species by microorganisms, viruses, helminths, yeasts and protozoans can have serious environmental, ecological and commercial implications. Not only are the birds themselves at risk, but there is also the potential of the infecting agent to spread to other animals, including humans.
The poultry industry is particularly vulnerable to significant economic losses due to the susceptibility of stock birds, and in particular newly-hatched stock birds, to rapidly spreading infections. In poultry birds, infection by Salmonella species, generally referred to as salmonellosis, is the most common form of infection causing high mortality rates. Many species of Salmonella also cause infection in humans and other animals and, hence, control of salmonellosis in poultry birds is of particular importance. Two of the most common species of Salmonella isolated from poultry birds are Salmonella typhimurium and Salmonella enteritidis. Both organisms contribute significantly to the outbreaks of salmonellosis and, in fact, S. enteritidis phage type 4 emerged as a significant threat to public health in Britain during the mid 1980's.
Chemotherapy and chemoprophylaxis have been used as forms of protective measures to combat poultry diseases such as salmonellosis. However, such measures are not always successful, are quite expensive, may lead to the development of drug resistance amongst infecting agents and are not necessarily acceptable to public health authorities for birds destined for human or animal consumption. For these and other related reasons, alternative forms of protection for avian species have been the subject of intense scientific research.
One form of protection proposed is the development of vaccines against poultry diseases. Live attenuated Salmonella vaccines have been shown to protect chickens (Cooper et al., Microb. Pathog., 9: 255-265, 1990; Hassan and Curtiss III, Res. Microbiol., 141: 839-850, 1990). Furthermore, Salmonella strains with a transposon inserted at or near aroA of the aromatic biosynthetic pathway have been shown to be attenuated, yet still capable of residing in tissue for sufficient time to stimulate an immune response (Stocker, Vaccine, 6: 141-145, 1988; U.S. Pat. No. 4,735,801; U.S. Pat. No. 5,210,035). Cooper, et al., Vaccine 10: 247-254, 1992, made two strains of S. enteritidisphage type 4 with mutations in the aroA gene. These mutant strains were used as a live oral vaccine against oral and intravenous challenge. The results presented by Cooper et al., supra, showed that the S. enteritidis vaccines were protective in chickens following oral ingestion.
Notwithstanding the purported efficacy of the live oral vaccines comprising Salmonella species in protecting chickens, newly-hatched birds are particularly susceptible to Salmonella infection and a high mortality rate immediately post-hatching can have serious economic consequences. Furthermore, administering live oral vaccines is not always convenient and difficulties may result in ensuring adequate doses are received by the birds. Another option, therefore, is to inoculate the birds when in ovo.
European Patent Application No 0 291 173 proposed the administration of a non-replicating immunogen designed to induce immunity in embryos prior to hatching. The method was said to be especially useful for immunizing birds against avian coccidiosis using a sporulated Eimeria tenella oocyst extract. U.S. Pat. No. 4,458,630 also teaches that birds can be immunized against Marek's disease by injecting eggs, prior to hatching, with a replicating viral vaccine. However, in both cases, inoculation was intra-embryo, such as into the yolk sac or chorion allantoic fluid. If bacteria were to be inoculated in such a manner, the bacteria would "blow" the egg, causing it to go rotten and causing the embryo to die.
There is a need, therefore, to improve the technology relating to in ovo vaccines so that avian species can be protected against challenge by virulent organisms such as virulent strains of Salmonella. In accordance with the present invention, the inventors have discovered that microorganisms rendered attenuated or avirulent may be inoculated into defined tissues in ovo and that birds upon hatching are protected from challenge with a corresponding wild-type microorganism. The method of the present invention also enables the use of the modified microorganisms to carry antigenic epitopes in the form of immunogens for other avian pathogens to induce immunity against those pathogens. The present invention represents a major breakthrough in protecting newly-hatched birds and offers significant commercial advantages to poultry management.